Xi-Ming Xu, Guang-Jin Yuan, Jun-Jian Deng, Hong-Ting Guo, Miao Xiang, Fang Yang, Wei Ge and Shi-You Chen

Wuhan, China

Inhibition of 12-lipoxygenase reduces proliferation and induces apoptosis of hepatocellular carcinoma cells in vitro and in vivo

Xi-Ming Xu, Guang-Jin Yuan, Jun-Jian Deng, Hong-Ting Guo, Miao Xiang, Fang Yang, Wei Ge and Shi-You Chen

Wuhan, China

BACKGROUND:12-lipoxygenase (12-LOX) has been reported to be an important gene in cancer cell proliferation and survival, and tumor metastasis. However, its role in hepatocellular carcinoma (HCC) cells remains unknown.

METHODS:Expression of 12-LOX was assessed in a diethylnitrosamine-induced rat HCC model, and in SMMC-7721, HepG2 and L-02 cells using immunohistochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR). GST-πand Ki-67 were determinedin vivoby immunohistochemical staining. Apoptosis was evaluated by TUNEL assay. Cell viability and apoptosis were determined by MTT assay and flow cytometry, respectively. Apoptosis-related proteins in SMMC-7721 and HepG2 cells were detected by Western blotting.

RESULTS:Immunohistochemical staining and RT-PCR showed that 12-LOX was over-expressed in rat HCC and two HCC cell lines, while the expression was inhibited by baicalein, a specific inhibitor of 12-LOX. Baicalein inhibited cell proliferation and induced apoptosis in rat HCC and both cell lines in a dose- and time-dependent manner. Ourin vivostudy demonstrated that baicalein also reduced neoplastic nodules. Mechanistically, baicalein reduced Bcl-2 protein expression coupled with a slight increase of the expression of Bax and activation of caspase-3. Furthermore, baicalein inhibited the activation of ERK-1/2 (phosphorylated). Interestingly, the effects of baicalein were reversed by 12(S)-HETE, a metabolite of 12-LOX.

CONCLUSIONS:Inhibition of 12-LOX leads to reduced numbers of HCC cells, partially caused by increased apoptosis. 12-LOX may be a potential molecular target for HCC prevention and treatment.

(Hepatobiliary Pancreat Dis Int 2012;11:193-202)

hepatocellular carcinoma; 12-lipoxygenase; proliferation; apoptosis

Introduction

Hepatocellular carcinoma (HCC) is a major health problem worldwide, and ranks third in the leading causes of cancer mortality.[1]Approximately 700 000 new cases are diagnosed each year.[2]The incidence of HCC has also increased in Europe and the United States over the past few years.[3,4]However, the choice of treatment, especially for advanced HCC, is limited, and the prognosis is poor. Preventive approaches, notably chemoprevention, are therefore sorely needed.

Eicosanoids, generated from arachidonic acid by either the cyclooxygenase or lipoxygenase (LOX) pathway, have been implicated in the pathogenesis of a variety of human diseases, including cancer. LOXs constitute a heterogeneous family of lipid-peroxidizing enzymes, which are categorized into 4 subfamilies based on their positional specificity for incorporation of oxygen in the substrate arachidonic acid: 5-LOX, 8-LOX, 12-LOX and 15-LOX.[5,6]Several studies have shown that 12-LOX is over-expressed in a variety of tumors including prostate, esophageal, kidney and bladder cancers,[7-10]and is involved in cancer cell proliferation and survival, and tumor metastasis.[11,12]However, the role of 12-LOX in HCC is unknown. In the present study, we assessed the expression of 12-LOX in HCCcell lines and investigated its effects on cell proliferation and apoptosis, as well as its underlying mechanisms of action using bothin vitroandin vivomodels.

Methods

Cell line cultures

Human hepatoma cell lines SMMC-7721, HepG2 and the normal liver cell line L-02 (Wuhan University Cell Center) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL streptomycin, and 100 U/mL penicillin in a humidified 5% CO2atmosphere at 37 ℃.

Animal model and treatment

Male Wistar rats (weighing 200 g) were obtained from the Experimental Animal Center of Wuhan University, and kept under specific pathogen-free conditions. After acclimation for 6 to 7 days, the animals received intraperitoneal injections of diethylnitrosamine (DEN, Sigma Chemical Co., St. Louis, MO) at 50 mg/kg body weight once a week for 16 weeks.[12]The rats were divided into two groups: DEN (n=8) and DEN/baicalein (n=8), in which daily baicalein (a specific 12-LOX inhibitor) at 250 mg/kg or vehicle was administered through gastric gavage. Three age-matched normal rats were used as controls. The animals were sacrificed by decapitation 2 weeks after the last intraperitoneal injection. Liver samples were snap-frozen and stored at -80 ℃ or fixed in 10% buffered formalin and embedded in paraffin. All animals were given humane care in compliance with institutional guidelines.

Histological evaluation

Liver samples, approximately 1.0×0.5×0.3 cm3, were processed for light microscopy. The processing consisted of fixing the specimens in 10% formaldehyde for 12-24 hours, embedding them in paraffin, cutting sections at 5 μm and staining the sections with hematoxylin and eosin. A pathologist did a blind evaluation of the sections.

Immunohistochemical or immunocytochemical staining for 12-LOX, GST-πand Ki-6712-LOX staining

Cells were grown on slides, and fixed in acetone for 10 minutes. Endogenous peroxidase was quenched with 3% H2O2in phosphate-buffered saline (PBS) for 10 minutes. Nonspecific binding sites were blocked by incubation with 10% normal goat serum for 20 minutes. The cells were then incubated with the primary antibody, rabbit anti-12-LOX polyclonal antibody (Santa Cruz, diluted 1:100), overnight at 4 ℃, followed by incubation with the second antibody, HRP (horseradish peroxide)-conjugated goat anti-rabbit IgG (Maixin Corp., diluted 1:150) at room temperature for 20 minutes. Staining was developed in freshly prepared diaminobenzidine solution (DAB, Sigma Co.) for 3-5 minutes, and then counterstained with hematoxylin, dehydrated, air dried, and mounted. The cells incubated with PBS instead of the primary antibody were used as negative controls.

GST-πand Ki-67 staining

Fixed liver sections were deparaffinized with xylene, hydrated through a graded series of alcohols, and incubated in a citrate buffer (pH 6.0) in a microwave oven for antigen retrieval. Similar procedures were then performed as above. Rabbit anti-GST-π polyclonal antibody (promab, diluted 1:100), rabbit anti-Ki-67 antibody (promab, diluted 1:100), and HRP-polymer goat anti-rabbit IgG were used as the primary or second antibodies. The staining area for GST-π was used to identify preneoplastic or neoplastic foci. The proliferative index was determined by Ki-67 staining using a procedure similar to that described above, and was defined as the percentage of immunoreactive cells in 1000 randomly selected cells.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis for 12-LOX mRNA expression

Total RNA was isolated from approximately 30 mg frozen liver tissue or 2×106SMMC-7721, HepG2 or L-02 cells using the TRIzol protocol (TRIzol reagent, Invitrogen) as suggested by the supplier. RNA concentration was quantified using a spectrophotometer at a wavelength of 260 nm. One microgram of total RNA was reverse-transcribed by adding 10 pmol oligo(dT)20 primer, 4 μL 5×RT buffer, 2 μL dNTP mixture (10 mmol/L, Fermentas), 10 units RNase inhibitor, and 1 μL ReverTra Ace-α-TM (TOYOBO Co., LTD) with a final volume of 20 μL at 42 ℃ for 20 minutes. The reverse transcriptase was heat-inactivated at 99 ℃ for 5 minutes and cooled on ice. The sequences for the sense and antisense primers were: human 12-LOX: 5'-CTT CCC GTG CTA CCG CTG-3' and 5'-TGG GGT TGG CAC CAT TGA G-3'; rat 12-LOX: 5'-TCT GAC CTC CCT GTA GAC C-3' and 5'-GGA ATT GGT ACC CAA AGA A-3'; GAPDH: 5'-ACC ACA GTC CAT GCC ATC AC-3' and 5'-TCC ACC ACC CTG TTG CTG TA-3'; β-actin: 5'-GAG AGG GAA ATC GTG CGT GAC-3' and 5'-CAT CTG CTG GAA GGT GGA CA-3'.

The sizes of their amplified fragment were 337bp, 226bp, 450bp and 452bp, respectively. Amplification was performed with 40 cycles with initial incubation at95 ℃ for 5 minutes and final extension at 72 ℃ for 5 minutes, each cycle of which consisted of denaturation for 30 seconds at 94 ℃, annealing for 30 seconds at 54 ℃ and extension for 30 seconds at 72 ℃. Following PCR, 10 μL samples of amplified products were resolved by electrophoresis in 1% agarose gel, and stained with ethidium bromide. The levels of 12-LOX expression were semiquantitatively evaluated using a Kodak ultraviolet imaging system and an image analysis system (Vilber Lourmat, France), and normalized to GAPDH or β-actin.

MTT assay

The MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide] colorimetric assay was used to determine the survival rate of cells. The cells were seeded at 1×104cells per well in 96-well plates and cultured in a humidified 5% CO2atmosphere at 37 ℃for 24 hours. Baicalein was added to the medium at the indicated times and concentrations. After incubation, the cells were washed with PBS. Then the cells were incubated with 1×MTT at 37 ℃ for 4 hours, and the absorbance at 450 nm was recorded. The survival rates of the cells were calculated according to the following equation: survival rate=[experimental absorbance value/control absorbance value]×100%.

Flow cytometry assay

Flow cytometry was used to determine apoptosis rates with propidium iodide (PI) staining. Cells were treated with baicalein at the indicated times and concentrations, and collected (about 2×106). The cells were trypsinized, washed with PBS and fixed in 75% ethanol. The fixed cells were washed with PBS, incubated with PI (50 μg/mL) containing 100 μg/mL RNase A for 30 minutes at 37 ℃, and analyzed on a FACScan flow cytometer (Beckman Coulter, USA). The percentage of the cells that had undergone apoptosis was assessed as the ratio of the fluorescent area smaller than the G0/G1 peak to the total area of fluorescence.

TUNEL assay

Forin situdetection of apoptotic cells, the TUNEL assay was performed using a commercial kit (Calbiochem TdT-FragELTMDNA Fragmentation Detection Kit). SMMC-7721 or HepG2 cells were plated on slides. After treatment with baicalein, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 minutes at 4 ℃. The cells were incubated with TdT equilibration buffer for 10-30 minutes, and then with the labeling reaction mixture consisting of TdT and biotinylated nucleotides for 90 minutes at room temperature. The reaction was terminated with stop buffer. Labeled DNA fragments were visualized by incubation with streptavidin-horseradish peroxidase conjugate for 30 minutes followed by color development with diaminobenzidine. Apoptotic cells containing labeled DNA fragments were identified by dark brown staining over the nuclei as visualized under a light microscope.

For tissue sections, deparaffinization in xylene and hydration through a graded series of alcohols were followed by permeabilization with proteinase K (2 mg/mL, 1:100 in 10 mmol/L Tris, pH 8) for 20 minutes at room temperature. Endogenous peroxidase was quenched with 30% hydrogen peroxide (1:10 in methanol) for 5 minutes. The sections were then stained with the same procedure as described above, and counterstained with hematoxylin. The apoptotic index (AI) was expressed as the number of positively stained cells per 100 hepatocytes.

Western blotting

SMMC-7721 or HepG2 cells were treated with baicalein or 12 (S)-HETE (a metabolite of 12-LOX) at the indicated concentrations for 24 hours, and collected (about 2×106) and washed in ice-cold PBS. The cells were lysed in 1 mL RIPA buffer for 30 minutes at 4 ℃. Lysates were centrifuged at 9000 rpm for 10 minutes, and the resulting supernatant was collected and stored at -20 ℃ until assay. The protein concentrations were assayed with the Bradford method.

Equivalent aliquots of proteins were separated by 10% SDS-PAGE, and transferred onto nitrocellulose filters. The filters were blocked with 5% nonfat dry milk in PBS for 2 hours at 37 ℃, washed with PBST (phosphatebuffered saline with Tween 20) and incubated with primary antibodies to caspase-3 (1:400, Santa), Bcl-2 (1:400, Santa), Bax (1:400, Santa), phospho-ERK1/2 (1:400, Santa), ERK1/2 (1:400, Santa), and β-actin (1:200, ProMab) at 4 ℃ overnight. After 4 washes with PBST, the filters were incubated with the secondary antibodies HRP-conjugated goat anti-mouse IgG (1:10 000 for caspase-3, Bcl-2, Bax, phospho-ERK1/2, ERK1/2, and 1:80 000 for β-actin) for 1 hour at room temperature. The immunoreactive proteins were detected using an enhanced chemiluminescent detection system.

Statistical analysis

Data were presented as mean±SD unless otherwise indicated. Differences between two or more groups were analyzed using the Mann-WhitneyUtest or the Kruskal-Wallis test. The statistical analyses were performed with SPSS 13.0. APvalue of less than 0.05 was considered statistically significant.

Results

Expression of 12-LOX in human hepatoma cells inhibited by baicalein

The expression of 12-LOX protein was analyzed in SMMC-7721, HepG2 and L-02 cells using immunocytochemistry. Moderately stronger staining was observed in SMMC-7721 and HepG2 cells, but no or faint staining in L-02 cells. The staining was mainly located in the cytoplasm. After treatment with 20 μmol/L baicalein (a specific 12-LOX inhibitor) for 48 hours, both SMMC-7721 and HepG2 cells showed reduced immunoreactivity (Fig. 1A). We then measured 12-LOX using Western blotting. There was extensive positivestaining in the SMMC-7721 and HepG2 cells. Treatment with 20 μmol/L baicalein for 48 hours resulted in loss of proteins (Fig. 1B).

Fig. 1. 12-LOX protein and mRNA expression in human hepatoma cells. A: 12-LOX protein expression analyzed by immunocytochemistry and Western blotting analysis. SMMC-7721, HepG2 or L-02 cells were incubated with 0 or 20 μmol/L baicalein for 48 hours, and then stained with 12-LOX antibodies. B: Expression of the indicated proteins was assayed by Western blotting. The bar graphs show the relative expression levels of indicated proteins after normalization to GAPDH. C, D: Expression of 12-LOX mRNA analyzed by RT-PCR. SMMC-7721 or HepG2 cells were treated with baicalein at 0, 5, 10, 20 and 40 μmol/L for 48 hours, or treated with 20 μmol/L baicalein for 0, 12, 24, 48 and 72 hours. RNA samples were extracted using the TRIzol reagents, followed by RT-PCR analysis. β-actin was used as internal control. The bar graphs show the relative expression levels of 12-LOX after normalization to β-actin. *: P<0.05, compared with untreated cells.

The expression of 12-LOX was then confirmed using RT-PCR with total RNA from both SMMC-7721 and HepG2 cells. Furthermore, the inhibition of 12-LOX expression by baicalein treatment was dose- and timedependent (Fig. 1 C, D).

Inhibition of growth of human hepatoma cells by 12-LOX

To investigate the effect of 12-LOX on HCC cell growth, we used the MTT assay. SMMC-7721 or HepG2 cells were treated with various concentrations of baicalein (5, 10, 20 and 40 μmol/L) for 48 hours, and their viability was analyzed. Baicalein treatment resulted in cell growth inhibition in a dose-dependent manner (Fig. 2A). Then, the cells were exposed to baicalein at 20 μmol/L and analyzed for cell number at different time points. We found that baicalein induced a decrease in cell growth which was also time-dependent (Fig. 2B). However, baicalein treatment of L-02 cells had no significant effect on cell growth.

Apoptosis of human hepatoma cells induced by 12-LOX

Fig. 2. Analysis of cell growth of SMMC-7721, HepG2 or L-02 cells treated with baicalein. A: Cells were treated with 0, 5, 10, 20 and 40 μmol/L baicalein for 48 hours. Cell survival rates were determined using MTT assay. B: Cells were incubated with 20 μmol/L of baicalein for 0, 12, 24, 48 and 72 hours. Cell survival rates were determined using MTT assay. *: P<0.05, compared with untreated cells.

The TdT-FragELTMDNA fragmentation detection assay was then used to evaluate apoptosis. Dark brown staining over the nuclei indicated apoptotic cells. Treatment with 20 μmol/L baicalein for 48 hours resulted in extensive positive staining in the SMMC-7721 and HepG2 cells, whereas no staining was observed in untreated cells or cells pretreated with 12-HETE (Fig. 3A). These positive-staining cells had sharply delineated masses or crescents of condensed chromatin, a signature of apoptotic cell death.

To further confirm induction of apoptosis by 12-LOX inhibition, flow cytometry was performed. Gradually increasing apoptosis of SMMC-7721 and HepG2 cells was observed after 48 hours with increasing concentrations of baicalein (0, 5, 10, 20 and 40 μmol/L) (Fig. 3B).

Expression of apoptosis-related proteins induced by 12-LOX but reversed by 12-HETE

Active caspase-3 expression is a key indicator of intracellular signaling of apoptosis, while Bcl-2 (antiapoptotic) and Bax (pro-apoptotic) are members of the Bcl-2 family, and are central regulators of apoptosis. Western blotting was used to detect the expression of active caspase-3, Bcl-2 and Bax protein levels. SMMC-7721 or HepG2 cells were treated with various concentrations of baicalein (0, 5, 10, 20 and 40 μmol/L) for 48 hours. The levels of active caspase-3increased gradually in a concentration-dependent manner after exposure to baicalein (Fig. 4). In addition, a marked downregulation of Bcl-2 protein and a slight upregulation of Bax protein were found.

Fig. 3. Analysis of apoptosis in SMMC-7721 and HepG2 cells treated with baicalein. A: Cells were treated with 0 or 20 μmol/L baicalein for 48 hours, and then stained with TUNEL. B: Cells were treated with 0, 5, 10, 20 and 40 μmol/L baicalein for 48 hours, followed by analysis with flow cytometry. The percentages of apoptotic cells are shown in the bar graphs. *: P<0.05, compared with untreated cells.

Fig. 4. Western blotting analysis of apoptosis-related proteins and ERK1/2 in SMMC-7721 (A and B) and HepG2 cells (C and D) treated with baicalein or 12-HETE. The cells were treated with 0, 5, 10, 20 and 40 μmol/L baicalein for 48 hours, or pretreated with 12(S)-HETE (0.1 μmol/L) for 3 hours followed by treatment with 20 μmol/L baicalein for 48 hours. Expression of the indicated proteins was assayed by Western blotting. The bar graphs show the relative expression levels of indicated proteins after normalization to GAPDH. P<0.05, compared with untreated (*) or 20 μmol/L baicalein-treated cells (#).

SMMC-7721 or HepG2 cells were pretreated with 12(S)-HETE (0.1 μmol/L) for 3 hours, and then treated with 20 μmol/L baicalein for 48 hours. The effects of baicalein on the above apoptosis-related proteins were reversed by the exogenous 12(S)-HETE (Fig. 4).

The ERK1/2 pathway blocked by 12-LOX

To understand whether the ERK1/2 pathway was affected by baicalein-induced 12-LOX inhibition, we then measured ERK1/2 and their active form using Western blotting. SMMC-7721 or HepG2 cells were treated with various concentrations of baicalein 0, 5, 10, 20 and 40 μmol/L) for 48 hours and total protein extracts were subjected to ERK1/2 evaluation. A concentrationdependent decrease in the levels of phosphorylated ERK1/2 was found; however, there was no change in the total ERK1/2 expression (Fig. 4). When the cells were pretreated with 12(S)-HETE (0.1 μmol/L) for 3 hours, the decrease of phosphorylated ERK1/2 mediated by baicalein (20 μmol/L) was reversed. Interestingly, the levels of activated ERK1/2 were even higher than those in the untreated cells, suggesting that 12(S)-HETE may activate phosphorylation of ERK1/2.

Platelet-type 12-LOX activated in rat HCC and blocked by baicalein

After 16 weeks of intraperitoneal injections of DEN, the rats developed nodules in the liver, which were confirmed histologically to be HCC (Fig. 5A). The mRNA expression of platelet-type 12-LOX was assessed by RT-PCR. 12-LOX expression levels were high in HCC samples compared with the very low levels in the control rats. Interestingly, a marked induction of platelet-type 12-LOX expression was found in the liver of DEN-induced rats, indicating that baicalein treatment blunted the induction of platelet-type 12-LOX expression.

HCC development inhibited by 12-LOX in rat

The liver in DEN-treated rats showed a distorted architecture with numerous neoplastic nodules, and baicalein treatment significantly reduced these nodules (Fig. 5A). GST-π is a sensitive marker for hepatic preneoplastic or neoplastic nodules. There were no GST-π-positive liver foci in control rats; however, extensive areas of GST-π-positive liver foci wereobserved in the DEN group. The GST-π-positive area was significantly reduced in the liver of rats treated with baicalein compared with the DEN group (Fig. 5B).

Fig. 5. Effects of baicalein on DEN-induced HCC in rats. Rats were subjected to 16 weeks of DEN administration and treated with baicalein or vehicle. A: Liver sections were stained with hematoxylin and eosin (HE), GST-π, Ki-67 or TUNEL. B: The bar graphs show the relative expression levels of GST-π, Ki-67 or TUNEL. C, D: RT-PCR analysis of 12-LOX mRNA in the liver of rats. β-actin was used as internal control. The bar graphs show the relative expression levels of 12-LOX after normalization to β-actin. P<0.05, compared with control (*) or DEN group (#).

Cell proliferation inhibited but apoptosis induced by 12-LOX in rat HCC

Since the nuclear antigen Ki-67 is a marker expressed only in proliferating cells, immunohistochemical staining for this marker was performed to evaluate proliferative activity. In the present study, positivelystained nuclei were scarce in the livers of the control rats, whereas numerous positively-stained nuclei were observed in the DEN group. Treatment with baicalein caused a marked reduction of the positive staining tothe extent that it was less than half of that in the DEN group (Fig. 5C), indicating inhibited cancer growth.

TUNEL assays were then performed to detectin situapoptosis. Extensive positive staining was observed in the DEN/baicalein group, whereas only a few positivelystained spots were seen in the control and DEN groups (Fig. 5D).

Discussion

12-LOXs are a family of isozymes that belong to the LOX superfamily, and catalyze the transformation of arachidonic acid into 12(S)-HPETE and 12(S)-HETE. At least three types of 12-LOX have been characterized, the platelet-type, the leukocyte-type, and the epidermal isoforms. The platelet-type 12-LOX is overexpressed in a variety of tumors including prostate, esophageal, kidney and bladder cancers,[7-10]and has been implicated in tumor promotion, progression and metastasis.[10-13]However, the role of 12-LOX in HCC remains unclear. In the present study, we investigated the expression of platelet-type 12-LOX in two human HCC cell lines: SMMC-7721 and HepG2. We found that the mRNA and protein levels of 12-LOX were detectable in both cell lines by immunocytochemistry and RT-PCR, but expression was not present in the normal liver cell line L-02. Several studies have reported that 12-LOX inhibitors exert antiproliferative effects and induce apoptosis in a variety of human cancers.[14-16]Consistent with other studies, our results showed that baicalein, a specific 12-LOX inhibitor, inhibited the growth of SMMC-7721 and HepG2 cells and induced apoptosis in a dose- and time-dependent manner. The expression of 12-LOX in the cells was also inhibited by baicalein, and the inhibition of the mRNA expression was shown to be concentration- and time-dependent. These results suggest that the inhibition of 12-LOX expression is a direct result of baicalein treatment in SMMC-7721 and HepG2 cells.

The mechanisms underlying the induction of apoptosis in HCC cells by a 12-LOX inhibitor were then investigated. The Bcl-2 family proteins consist of antiapoptotic (Bcl-2, Bcl-XL) and pro-apoptotic members (Bax, Bcl-XS, Bad, Bak, Bik), which are the major regulators of apoptosis.[17]The ratios between these antiapoptotic and pro-apoptotic proteins control cell fate. Several studies have shown that 12-LOX inhibition by baicalein or N-benzyl-N-hydroxy-5-phenylpentamide leads to decreased Bcl-2 and increased or constant Bax expression in prostate and gastric cancer cells.[14,18]Similar to other studies, we also found a dose-dependent decrease in Bcl-2 protein expression in both HCC cell lines after treatment with baicalein, and the reduction was associated with a slight increase of Bax expression, which resulted in a shift in the Bcl-2/Bax ratio favoring apoptosis. Caspase-3 is the executioner of apoptosis, and its active form is considered a key indicator of apoptosis.[19]In our study, a dose-dependent increase in the active form of caspase-3 was found in HCC cell lines after treatment with baicalein. To confirm the role of 12-LOX inhibition in the apoptosis of HCC cells, the cells were pretreated with a metabolite of 12-LOX, 12(S)-HETE. The results showed that all the effects of baicalein were reversed. These data demonstrate that 12-LOX is involved in HCC cell survival, and inhibition of 12-LOX induces apoptosis through altering the expression of Bcl-2 and Bax while activating caspase-3.

ERK1/2 are members of the mitogen-activated protein kinase family, and have been implicated in diverse cellular processes including proliferation, differentiation and survival.[20-23]They have been shown to be activated in human HCC, and may play an important role in multistep hepatocarcinogenesis, especially in the progression of HCC.[24]A number of growth factors and cytokines can activate ERK1/2.[25]12(S)-HETE has been reported to activate ERK1/2 in pancreatic cancer cells, which are involved in 12(S)-HETE-induced cell proliferation.[26]In the present study, we found that baicalein down-regulated active (phosphorylated) ERK1/2 in a dose-dependent manner in both SMMC-7721 and HepG2 cells, but the effect was reversed by 12(S)-HETE. These findings suggest that inhibition of 12-LOX blocks ERK1/2 activation, leading to the arrest of cell proliferation.

In view of the above findings,in vivostudies were carried out to determine the effect of 12-LOX inhibition on chemically-induced HCC in rats. The gene expression of platelet-type 12-LOX in the liver of rats with HCC induced by DEN was markedly increased compared with control rats. However, treatment with baicalein significantly blunted this increase. These results were consistent with ourin vitrostudies in cultured cell lines, suggesting that baicalein inhibits not only the enzymatic activity of 12-LOX, but also its expression, which may be an important preventive mechanism of baicalein against HCC. The neoplastic nodules identified by GST-π staining in the liver were also significantly reduced as compared with DEN-induced model rats.[27]These results indicate that 12-LOX participates in the pathogenesis of HCC, and the inhibition of 12-LOX by baicalein may block the development of this tumor, which is consistent with the previous study that quercetin and nordihydroguiaretic acid as nonspecific lipoxygenase inhibitors inhibit the development ofhepatic preneoplastic and neoplastic lesions in Fischer 344 male rats fed a choline-deficient, L-amino aciddefined diet.[28]Similar to the abovein vitroresults, treatment with baicalein also caused a marked reduction in cell proliferation and the induction of apoptosis in rat HCC, evidenced by Ki-67 and TUNEL staining. These results demonstrate that the blockade of HCC development by a 12-LOX inhibitor is attributable to its inhibition of proliferation and induction of apoptosis in HCC cells.

In summary, our study demonstrated that 12-LOX contributes to the pathogenesis of HCC. Inhibition of 12-LOX causes inhibition of cell proliferation and induction of apoptosis in HCC cellsin vitroandin vivo, the mechanism of which may be associated with blockade of ERK1/2 activation and altered expression of Bcl-2 and Bax. Therefore, 12-LOX is a potential molecular target for HCC prevention and treatment.

Contributors:XXM proposed the study. YGJ carried out immunohistochemical and cytochemical staining experiments. DJJ carried out the proliferation, apoptosis assay and animal model and treatment. GHT carried out TUNEL and flow cytometry assays. XM carried out the cell culture and RT-PCR. GW carried out MTT assays and Western blotting analysis. YF carried out histological evaluation and participated in the design of the study. CSY performed the statistical analysis. All authors read and approved the final manuscript. XXM is the guarantor.

Funding:This study was supported by grants from the National Natural Science Foundation of China (81000998), the Natural Science Foundation of Hubei Province, China (2007ABA248) and the Foundation of the Ministry of Education of China for New Teachers (20090141120003).

Ethical approval:All animals were given humane care in compliance with institutional guidelines.

Competing interest:The authors do not choose to declare any conflict of interest related directly or indirectly to the subject of this article.

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March 30, 2011

Accepted after revision July 19, 2011

Retraction

10.1016/S1499-3872(12)60148-9)

Author Affiliations: Cancer Center, Renmin Hospital of Wuhan University, Wuhan 430060, China (Xu XM, Deng JJ, Guo HT, Xiang M and Ge W); Cancer Center, the 82nd Hospital of the Chinese PLA, Huai'an 223001, China (Yuan GJ); Department of Physiology, Medical College of Wuhan University, Wuhan 430071, China (Yang F); Department of Physiology & Pharmacology, University of Georgia, Athens, GA 30602, USA (Chen SY)

Xi-Ming Xu, MD, PhD, Cancer Center, Renmin Hospital of Wuhan University, Wuhan 430060, China (Tel: 86-27-88041911; Fax: 86-27-88042292; Email: whuxxm@yahoo.com)

? 2012, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(12)60147-7

It has come to the attention of the editors that the paper (Fulminant liver failure models with subsequent encephalopathy in the mouse. Baine AMT, Hori T, Chen F, Gardner LB, Uemoto S and Nguyen JH. Hepatobiliary Pancreat Dis Int 2011;10:611-619.) was published in Annals of Gastroenterology in 2011 before its appearance in our journal. Therefore we issue a retraction notice for readers to discourage citations of the paper.